The present invention relates to a method of diagnostic of the diseases caused by mutations in the LKB1 gene and to an diagnostic reagent and a therapeutic preparation for said diseases.
Peutz-Jeghers (PJ) syndrome [MIM 175200] is an autosomal dominantly inherited disorder characterized by melanotic pigmentation of lips, perioral and buccal regions and by benign, hamartomatous and adenomatous types of multiple gastrointestinal polyps. Patients with this syndrome are known to frequently develop benign or malignant neoplasms in the gastrointestinal tract, pancreas, ovaries, testis, breast and uterus. In particular, small benign tumors frequently occur in the ovary and are developed as multifocal, symmetrical germinal-cord structures with annular tubules. Then, they progress into granulosa cancer and result in ambisexual precocity in girls. It was found that multifocal germinal cord cancer in boys, though with low frequency, results in gynecomasty and feminization due to over-production of estrogen. Defects in the gene responsible for Peutz-Jeghers syndrome (PJ gene) appear to predispose to a wide spectrum of neoplastic diseases. In fact, 50% of the carriers with a defect in one of PJ allele are known to develop cancer by the age of 60 (Giardiello, F. M. et al. Increased risk of cancer in Peutz-Jeghers syndrome. N. Engl. J. Med. 316,1511-1514 (1987); Spigelman, A. D., Murday, V. and Phillips, R. K. Cancer and Peutz-Jeghers syndrome. Gut 30, 1588-1590 (1989). [MIM 175200]). It is believed that loss or inactivation of the PJ gene product results in disruption of the fundamental growth control mechanism within somatic cells that have potential high proliferative capacity, which triggers the growth of benign hamartomatous polyps some of which turn into malignant tumor cells after further genetic alteration.
Recently, it was reported that PJ gene was mapped to chromosome 19p13.3 by linkage analysis in 12 families of PJ syndrome with a multipoint lod score of 7.00 at the microsatellite genetic marker D19S886 (Hemminki, A. et al. Localization of a susceptibility locus for PJ syndrome to 19p using comparative genomic hybridization and targeted linkage analysis. Nat. Genet. 15 (1), 87-90 (1997)). A similar linkage between PJ gene and the genetic marker D19S886 was reported in a second study investigating five other families as well (with a multipoint lod score of 7.52) (Amos, C. I. et al. Fine mapping of a genetic locus for PJ syndrome on chromosome 19p. Cancer Res. 57, 3653-3656 (1997)). D19S565 was first known as a genetic marker proximal to the PJ gene, which causes recombination with the gene. Thereafter, the genetic marker D19S878 was found to be located more proximal to the gene. These two makers, therefore, were thought to define the proximal border of the PJ candidate region. In both linkage studies, no recombination was observed between the marker D19S886 and PJ gene, indicating that they are located in a narrow interval.
An objective of the present invention is to identify the gene responsible for Peutz-Jeghers syndrome and to provide a method for diagnosing diseases resulting from a mutation in this gene, a diagnostic reagent and a therapeutic preparation for the diseases.
To identifiy the gene for PJ syndrome, the present inventors made a continuous cosmid contig and a restriction map for the region extending from 1.5 Mb within chromosome 19p13.3 which includes the PJ gene. EST database search for the genes mapped in this region was then performed, and the precise locations of these genes were determined. The inventors evaluated biological information of a number of genes thus found and selected several potent candidates of the PJ gene. Mutation analysis of these candidate genes in DNAs from patients with PJ syndrome revealed that one of the candidate genes, xe2x80x9cLKB1,xe2x80x9d was specifically mutated in the patients with PJ syndrome. Thus, the inventors"" intense investigations successfully identified the gene responsible for PJ syndrome for the first time. Based on this finding, the inventors found that diseases caused by mutations in the LKB1 gene can be diagnosed and treated by utilizing the LKB1 gene, primers and probes based on the sequence thereof, LKB1 protein and antibodies that bind to LKB1 protein.
As described above, the present invention relates to a method of diagnosing diseases caused by mutation in the LKB1 gene, the gene responsible for PJ syndrome, and to a diagnostic reagent and a therapeutic preparation for the diseases. More specifically, the present invention relates to:
(1) a primer DNA used for diagnosing a disease caused by mutation in the LKB1 gene, the primer DNA comprising a nucleotide sequence containing at least a portion of any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(2) the primer DNA according to (1), wherein the primer DNA has a nucleotide sequence corresponding to any one of the nucleotide sequences shown in SEQ ID NOs: 7 to 30;
(3) the primer DNA according to (1) or (2), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(4) a probe DNA used for diagnosing a disease caused by mutation in the LKB1 gene, the probe DNA comprising a nucleotide sequence containing at least a portion of any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 4;
(5) the probe DNA according to (4), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(6) a therapeutic preparation for a disease caused by mutation in the LKB1 gene, the preparation comprising the LKB1 gene as an active ingredient;
(7) a therapeutic preparation for a disease caused by mutation in the LKB1 gene, the preparation comprising the LKB1 protein an active ingredient;
(8) a therapeutic preparation for a disease caused by mutation in the LKB1 gene, the preparation comprising a compound that enhances the activity of LKB1 protein as an active ingredient;
(9) the therapeutic preparation according to (6) to (8), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(10) a reagent for diagnostic of a disease caused by mutation in the LKB1 gene, the reagent comprising an antibody that binds to the LKB1 protein as an active ingredient;
(11) the reagent according to (10), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome;
(12) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising detecting mutation in the LKB1 gene;
(13) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a DNA sample from a patient;
(b) amplifying the DNA using the primer DNA according to (1);
(c) cleaving the amplified DNA;
(d) fractionating the DNA fragments according to their size;
(e) hybridizing the probe DNA according to (4) with the fractionated DNA fragments; and
(f) comparing the size of the DNA fragment thus detected to that from a control of a healthy subject;
(14) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a RNA sample from a patient;
(b) fractionating the RNA sample depending on its size;
(c) hybridizing the probe DNA according to (4) with the RNA thus fractionated;
(d) comparing the size of the RNA thus detected to that from a control of a healthy subject;
(15) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a DNA sample from a patient;
(b) amplifying the DNA using the primer DNA according to (1)
(c) separating the amplified DNA into single stranded DNA;
(d) fractionating the separated single stranded DNA on a non-denatured gel;
(e) comparing the mobility of the single stranded DNA separated on the non-denatured gel to that of a control of a healthy subject;
(16) a method of diagnosing a disease caused by mutation in the LKB1 gene, the method comprising the steps of:
(a) preparing a DNA sample from a patient;
(b) amplifying the DNA using the primer DNA according to (1);
(c) fractionating the amplified DNA on the DNA denatured gradient gel;
(d) comparing the mobility of the fractionated DNA on the gel to that of a control of a healthy subject;
(17) the method according to any one of (12) to (16), wherein the disease caused by mutation in the LKB1 gene is Peutz-Jeghers syndrome.
The present invention was made based on the inventors, findings that Peutz-Jeghers syndrome is caused by a mutation in the gene called xe2x80x9cLKB1xe2x80x9d. The present invention primarily relates to the use of polynucleotides containing at least a portion of the nucleotide sequence corresponding to the genomic DNA coding LKB1 (including intron, promoter, and enhancer regions as well as exon regions), and said polynucleotides for diagnosing diseases resulting from mutations in the LKB1 gene. The genomic DNA regions of LKB1 are shown in the SEQ ID: 1 to 4. The sequences shown in SEQ ID: 1 to 4 correspond to 5xe2x80x2 upstream region, exon 1 and intron 1 (a part) region, exons 2 to 8 and introns 1 (a part) to 8 (a part) region, and intron 8 (a part) and exon 9 region of the LKB1 gene, respectively.
Nucleotide sequences containing a portion of these regions can be used as primers or probes to diagnose the diseases resulting from mutations in the LKB1 gene. A nucleotide used as a primer is typically 15 to 100 bp, preferably 17 to 30 bp. Any primer can be used as long as it amplifies at least a portion of the LKB1 gene or regions regulating the gene expression. Such regions include, for example, exon, intron, promoter, and enhancer regions of the LKB1 gene. On the other hand, nucleotides used as a probe typically have a sequence of at least 15 bp or more when the nucleotides are synthetic oligonucleotides. Double stranded DNA obtained from a clone into which a vector such as plasmid DNA is incorporated can be used as a probe. For a region utilized as a probe, any part of the LKB1 gene or region regulating the expression thereof can be used. Such regions include, for example, exon, intron, promoter and enhancer regions of the LKB1 gene. When used as a probe, oligonucleotide or double stranded DNA is used after adequately labeling. Labeling methods, for example, include labeling of the 5xe2x80x2-terminus by phosphorylating with 32P using T4 polynucleotide kinase, and labeling by incorporation of substrate bases labeled with isotopes such as 32P, fluorochrome or biotin using DNA polymerases such as Klenow enzyme and primers such as random hexamer oligonucletides (random-primer method).
The diseases detectable using these nucleotides, which are caused by mutations in the LKB1 gene are not limited to Peutz-Jeghers syndrome. Any disease caused by mutations in the LKB1 gene is included. PTEN and APC genes were discovered as causative genes for Cowden""s disease, which is one of hereditary cancers, and Familial Adenomatous polyposis (FAP), respectively. Both genes proved to be mutated with high frequency in the non-hereditary common cancers. PTEN and APC genes function to control cell proliferation in the normal tissues, and it is believed that a critical step in tumorigenesis occurs when these genes mutate and lose their functions resulting in cells escaping from the regulation of these genes. Similar to PTEN and APC genes, a mutation in the LKB1 gene possibly plays a part in common tumorigenesis.
The diagnostic method of the diseases caused by mutations in the LKB1 gene in the present invention features detection of mutations in the LKB1 gene. In the present invention, by the term xe2x80x9cthe diagnosis of the diseases caused by mutations in the LKB1 genexe2x80x9d is meant not only the testing of patients who have developed the particular symptoms resulting from a mutation in the LKB1 gene, but also testing a mutation in the LKB1 gene for determining whether the subject has a predisposition to the particular disease resulting from a mutation in the LKB1 gene. A mutation in one of allele of the LKB1 gene is thought to largely increase the risk for a particular disease caused by mutations in the LKB1 gene even if the symptom has not apparently been developed. The present invention also includes a diagnostic method for identifying patients who have a mutation in one of allele of the LKB1 gene (carriers). xe2x80x9cDetection of a mutation in the LKB1 genexe2x80x9d in the present invention includes detection in DNA, RNA and protein.
One embodiment of the diagnostic method of the present invention is a method of directly determining the nucleotide sequence of the LKB1 gene of a patient. For example, sequencing is performed after amplifying the whole or a partial sequence of the LKB1 gene of a patient using a technique such as PCR (Polymerase Chain Reaction), using the nucleotides described above as primers, and the DNA isolated from a patient who is suspected of being afflicted with a disease caused by a mutation in the LKB1 gene, as template. The sequence thus determined can be used to diagnose the diseases caused by mutations in the LKB1 gene by comparing it to the sequence of the LKB1 gene from healthy subjects.
In addition to the methods as described above in which DNA from a patient is directly sequenced, different methods can be used as diagnostic methods of the present invention. One of such embodiments comprises the steps of (a) preparing DNA samples from patients, (b) amplifying the DNA from the patients using the primer DNA of the present invention, (c) separating the amplified DNA into the single stranded DNA, (d) fractionating the single stranded DNA separated on non-denaturing gel, and (e) comparing the mobility of the single stranded DNA separated on the gel to that of a control of ordinary person.
Such methods include PCR-SSCP (single-strand conformation polymorphism; polymorphism of single-stranded DNA in higher-order structure) method (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11. Genomics. 1992 Jan. 1; 12(1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1991 Aug. 1; 6(8): 1313-1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr. 1; 4(5): 275-282.). Having the advantage of relatively simple manipulation and smaller sample volume requirement, this method is particularly suitable for screening a large number of DNA samples. The principles of the method are as follows: when double stranded DNA fragments are denatured into single stranded DNA, each single stranded DNA forms an original higher-order structure peculiar to its nucleotide sequence; these denatured DNA, even if they are complementary to each other and have the same chain-length, migrate to distinct positions according to their conformation when electrophoresed on a non-denaturing polyacrylamide gel; the conformations of these single stranded DNA are altered by substitution of a single nucleotide and the substituted DNA migrate with different mobility by electrophoresis on polyacrylamide gel; and thus, detection of the alteration in the mobility enables the detection of mutations such as point mutation, deletion or insertion in the DNA fragment of the interest.
In the PCR-SSCP method, the whole or a portion of the LKB1 gene is initially amplified using a technique such as PCR. A nucleotide sequence ranging in length from about 200 to 400 bp is typically preferred as a fragment to be amplified. Portions to be amplified include an exon, intron, promoter and enhancer of the LKB1 gene. PCR can be performed under the conventional conditions (for example, the conditions used for amplification of each exons using the primers shown in example 5). On amplifying the gene fragment by PCR, DNA fragments to be synthesized in the PCR reaction may be labeled with primers labeled with an isotope such as 32P, fluorochrome or biotin, or by adding substrate nucleotides labeled with an isotope such as 32P, fluorochrome or biotin in the PCR reaction solution. The said DNA fragments may also be labeled by adding substrate nucleotides labeled with an isotope such as 32P, fluorochrome or biotin to the synthesized DNA fragments by using Klenow enzyme after the PCR reaction. The labeled DNA fragments thus obtained are denatured by heat, for example, and electrophoresed on a polyacrylamide gel without any denaturing agent, such as urea. As to the electrophoresis, conditions for fractionating DNA fragments can be improved by adding an appropriate amount of glycerol (5 to 10%) to polyacrylamide. Conditions for electrophoresis vary depending on the nature of each DNA fragment. Electrophoresis is typically performed at room temperature (20 to 25xc2x0 C.), but when preferable fractionation is not obtained, in the temperature ranging from 4 to 30xc2x0 C., it is better to test the optimal mobility to determine the temperature which gives the most desirable mobility. After the electrophoresis, mobility of the DNA fragment is detected and analyzed by autoradiography using X-ray films and by scanner for detection of fluorescence. When the bands with different mobility are detected, those bands are directly dissected from the gel, re-amplified by PCR, and subjected to direct sequencing to confirm the mutation. In case where DNA fragments synthesized by PCR are not labeled, the bands of said DNA fragments may be detected using staining techniques such as ethidium bromide or silver staining.
Another embodiment of the diagnostic methods of the present invention comprises the steps of (a) preparing a DNA sample from a patient, (b) amplifying the DNA derived from the patient using the DNA primers of the present invention, (c) cleaving the DNA thus amplified, (d) fractionating the DNA fragments depending on their size, (e) hybridizing the DNA fragments thus fractionated with the probe DNA of the present invention, and (f) comparing the length of the DNA fragment thus detected to that from healthy subjects.
These methods include the methods utilizing restriction fragment length polymorphism (RFLP) and PCR-RFLP method. These methods are based on the principle that when a mutation has occurred in the recognition site for a restriction enzyme or when insertion or deletion of bases has occurred in the DNA fragments generated by treatment with a restriction enzyme, the length of those fragments treated with the restriction enzyme generally deviates from that of normal subjects. In fact, for Peutz-Jeghers syndrome, in the LKB1 gene of the four patients shown as samples, D, B, MA and FA, with Peutz-Jeghers syndrome, acquisition of ScaI site, elimination of AhdI, RsaI, and BsrBI site had occurred, respectively (Table 3). Therefore, these mutations can be detected as the differences of bands"" mobility after electrophoresis, which was performed after the portions containing these mutations are amplified by PCR and then treated with the restriction enzymes mentioned above. Alternatively, the mutations can be detected by subjecting DNA from patients to southern blotting using the probe DNA of the present invention, after the DNA is treated with these restriction enzymes and electrophoresed. Restriction enzymes used other than those mentioned above are properly selected depending on each mutation. In this method, besides detecting the mutations via restriction enzyme treatment of genomic DNA prepared from patients, cDNA prepared by the treatment of RNA prepared from patients with reverse transcriptase is directly treated with the restriction enzyme and then can be used for southern blotting to detect mutations. This cDNA can also be used as a template for PCR, and the amplified product thereof (the whole or a portion of the LKB1 gene) can be digested with a restriction enzyme and then electrophoresed to detect mutations shown as difference of mobility of the DNA fragments.
RNA prepared from patients may be used for the detection instead of DNA. Such a method comprises the steps of (a) preparing an RNA sample from a patient, (b) fractionating the prepared RNA depending on the size, (c) hybridizing the RNA thus fractionated to the DNA probe of the present invention, and (d) comparing the size of the RNA fragment thus detected to that from normal subjects. Specifically, RNA prepared from a patient is electrophoresed and subjected to northern blotting to detect the difference of mobility.
Other embodiments of the diagnostic methods of the present invention comprise the steps of (a) preparing a DNA sample from a patient, (b) amplifying the DNA derived from the patient using the primers of the present invention, (c) fractionating the amplified DNA by electrophoresis on the denaturing gradient gel, (d) comparing the mobility of the DNA on the gel fractionated to that from normal subjects.
A similar method includes denaturant gradient gel electrophoresis (DGGE) method. In this method, the whole or a portion of the LKB1 gene is amplified by PCR using primers of the present invention, for example, and electrophoresed on the polyacrylamide gel in which the concentration of a denaturant such as urea gradually increases as the DNA migrate through the gel. The mobility of the DNA is compared to that of normal subjects. If a DNA fragment contains a mutation, it comes to be melted into single stranded DNA in the point of lower denaturant concentration and is remarkably retarded in mobility, and detection of such difference of mobility allows detection of a mutation.
Besides these methods, allele specific oligonucleotide (ASO) hybridization method can be used for the purpose of only detecting only a mutation in a specific position. In this method, an oligonucleotide containing sequence supposed to have a mutation is synthesized and hybridized with a DNA sample. If the DNA sample has a mutation, the efficiency of hybridization decreases, and this decrease is detected using techniques such as southern blotting and the method of utilizing the florescence quenching property of specific florescent reagents, which are quenched when intercalated into the gap between the hybrids.
Ribonuclease A mismatch cleavage may also be used for the detection. In this method, the whole or a portion of the LKB1 gene is amplified by PCR, for example, and the amplified DNA is subjected to hybridization with labeled RNA prepared from LKB1 cDNA or and such, which is inserted into a plasmid vector and such. This hybrid forms single stranded structures at the sites of mutation, and said sites may be cleaved by ribonuclease A and then detected using some method such as autoradiography. Presence of mutations, can be thus detected.
The present invention relates to a diagnostic reagent for diseases caused by mutations in the LKB1 gene, wherein the reagent comprises, as an active ingredient, an antibody, which binds to the LKB1 protein. Antibodies, which bind to LKB1 protein, can be prepared according to methods well known in the art. As for polyclonal antibodies, for example, a small animal such as a rabbit is immunized with the LKB1 protein (a natural protein as well as a recombinant KLB1 protein expressed in suitable host cells (E. coli, yeast or mammalian cells), such as LKB1 protein expressed as a fusion protein with GST in E. coli) or its partial peptide (for example, peptide composed of amino acid sequence shown in the SEQ ID: 31 or 34) to obtain antiserum, which then can be purified and prepared using a method such as ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity columns coupled with LKB1 protein or synthetic peptide. For monoclonal antibodies, for example, a small animal such as a mouse is immunized with LKB1 protein or a part of the peptide, and then dissected to remove the spleen. The spleen is homogenized to isolate the cell fraction. These cells are fused with mouse myeloma cells and a reagent such as polyethylene glycol to create fusion cells (hybridomas). From the hybridoma cells thus generated, an appropriate clone which produces an antibody which binds to LKB1 protein is selected. Subsequently, the hybridoma cells thus obtained are intraperitoneally transferred into a mouse, ascites is harvested from the same mouse, and the monoclonal antibody thus obtained can be prepared by purifying by means of, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity columns coupled with LKB1 protein or synthetic peptide.
When used as a reagent, the antibody is, if necessary, mixed with sterile water, physiological saline, vegetable oils, surface active agent, lipids, solubility increasing agent, stabilizers (e.g. BSA and gelatin), and preservatives and such. In a test using said antibody, a tissue or cells from a patient are stained using a method such as enzyme-labeled or fluorescence-labeled antibody technique to detect deficiency, aberrant accumulation, or unusual intracellular distribution of LKB1 protein. Alternatively, the protein, which is fractionated from cell extracts prepared from a tissue or cells from patients with Peutz-Jeghers syndrome using a method such as SDS-PAGE, is transferred onto a membrane such as nitrocellulose or PVDF, and detected using a staining method such as enzyme-labeled technique above (western blotting, immunoblotting).
By constructing a detailed physical map of 19p13.3 region, the inventors revealed that the disease-related gene LKB1 for Peutz-Jeghers syndrome is located in the close proximity of a microsatellite marker D19S886, i.e. their distance on chromosome is about 190 kb. Therefore, the loss of heterozygosity (LOH) test utilizing D19S886 marker may effectively serve as a test for diagnosing various diseases based on mutations in the LKB1 gene.
The present invention also relates to a therapeutic preparation for diseases caused by mutations in the LKB1 gene. In one embodiment, it comprises the LKB1 gene as an active ingredient. When the LKB1 gene is used as a therapeutic preparation, the whole or a portion of the genomic LKB1 DNA, or the LKB1 cDNA (SEQ ID: 5) is incorporated into an appropriate vector, such as adenovirus vector, adenoassociated virus vector, retrovirus vector, and plasmid DNA, and administered orally, intravenously, or topically to the patient. As a method of administration, ex vivo administration can be used as well as in vivo administration. In administration of a drug, enclosing the gene into a liposome generated by micellization of phoshpholipids can enhance the mobility and intake of the gene into the tissue. Alternatively, cationic lipids may be added to form a complex with DNA, which can enhance the mobility and intake of the gene into the tissue. Using these methods, the LKB1 gene mutated in the patient can be substituted by a normal gene, or the normal gene can be additionally administered to the patient, resulting in the possible treatment of a disease caused by a mutation in LKB1 gene.
Another embodiment of a therapeutic preparation for the diseases caused by mutations in the LKB1 gene, comprises the LKB1 protein as an active ingredient. The LKB1 protein may be prepared as a natural protein or a recombinant protein by utilizing a recombinant DNA technology. The amino acid sequence of the LKB1 protein is shown in SEQ ID: 6. A natural protein may be isolated using well-known methods. For example, it can be isolated from the cultured cells of the testis, fetal liver or K562 cell, in which the LKB1 protein is expressed at a high level, and by affinity column chromatography using an antibody against a partial peptide of the LKB1 protein described in Example 7. On the other hand, a recombinant protein can be prepared by culturing cells transformed by DNA (SEQ ID: 5) encoding LKB1 protein. Cells that can be used to produce a recombinant protein include mammalian cells, such as COS, CHO, and NIH3T3 cells; insect cells, such as Sf9 cells; yeast; and E. coli. The vectors suitable for expressing a recombinant protein intracellularly depend on the host cells, for example, pcDNA3 (Invitrogen) or pEF-BOS (Nucleic Acids. Res. 1990, 18(17), p.5322) vector is used for mammalian cells; xe2x80x9cBAC-to-BAC baculovirus expression systemxe2x80x9d (GIBCO BRL) for insect cells; xe2x80x9cPichia Expression Kitxe2x80x9d (Invitrogen) for yeast; and pGEX-5X-1 (Pharmacia) and xe2x80x9cQIAexpress systemxe2x80x9d (Quiagen) for E. coil. The vectors can be introduced into the host cells by a well-known method such as the calcium phosphate method, DEAE dextran method, the method using cationic liposome DOTAP (Boehringer Mannheim) or SuperFect (Quiagen), electroporation, and calcium chloride. A recombinant protein thus obtained can be purified using a conventional technique, for example, the method described in xe2x80x9cThe Qiaexpressionist handbook, Quiagen, Hilden, Germanyxe2x80x9d.
When the LKB1 protein obtained is used as a therapeutic preparation for diseases caused by mutations in the LKB1 gene, the LKB1 protein can be directly administered, or can be given after formulating by a well-known pharmaceutical process. For example, formulations may be administered in proper combination with a pharmaceutically acceptable carrier or medium, such as sterile water, physiological saline, vegetable oils, surfactants, lipids, dissolving adjuvants, stabilizers, or preservatives. While the dosage for administration differs depending on various factors, such as weight, age, and health conditions, or the method of administration, a person skilled in the art will be able to advantageously select the appropriate dosage. Typically, the dosage is in the range of 0.01 to 1000 mg/kg. Administration can be conducted, for example, orally, intravenously, intramuscularly or subcutaneously.
A person skilled in the art can easily carry out substitution, deletion, addition and/or insertion of amino acids in the amino acid sequence of the LKB1 protein for the purpose of improving the activity and stability of the drug of the present invention, utilizing a well-known method, such as PCR-based site-directed mutagenesis (GIBCO-BRL, Gaithersburg, Md.), site-directed mutagenesis using oligonucleotides (Kramer, W. and Fritz, H J (1987) Methods in Enzymol., 154:350-367), the Kunkel""s method (Methods Enzymol. 85, 2763-2766 (1988)). A similar altered LKB1 protein can also be used as a therapeutic preparation of the present invention.
Another embodiment of a therapeutic preparation for diseases caused by mutations in the LKB1 gene, the therapeutic preparation comprises a compound, which enhances the activity of the LKB1 protein, as an active ingredient. The LKB1 gene encodes a serine threonine kinase that shows as high as 82% homology with XEEK1 serine threonine kinase from Xenopus. A collapse of the serine threonine kinase activity of the LKB1 protein is closely associated with the emergence of diseases resulting from mutations in the LKB1 gene. Therefore, it is envisaged that enhancement of the said serine threonine kinase activity will serve to treat diseases caused by mutations in the LKB1 gene.
A screening method for a compound that enhances the activity of the LKB1 protein is as follows. For example, LKB1 proteins expressed in E. coli as a fusion protein with GST, or those expressed in mammalian or insect cells, are used to determine kinase activities of these proteins in the presence of the test compound, to select a compound that enhances the activity of the LKB1 protein.
More specifically, for example, phosphorylation activity of a substrate protein for the LKB1 protein, or autophosphorylation activity of the LKB1 protein may be determined by measuring the transfer of 32P from [xcex3-32P]ATP to the substrate in an appropriate reaction solution (e.g. 50 mM Tris-HCl, pH 7.2, 1 mM dithiothreitol (DTT), 10 mM MgCl2, 10 mM MnCl2, and so on), using a device, such as a liquid scintillation counter, and thus a compound which enhances the activity of LIB1 protein can be isolated by selecting a compound that increass the 32P transfer level. Like the LKB1 protein used as therapeutic preparation as described above, the isolated compound may be formulated using well-known pharmaceutical processes to be administered for treatment of a disease. Typically, the dosage is in the range of 0.01 to 1000 mg/kg.
In addition to the methods as described above, a method utilizing the regions regulating the LKB1 gene expression, or a factor binding to the gene can be used for treatment of the diseases caused by mutations in the LKB1 gene. The present invention has revealed the structure of the LKB1 gene and the 5xe2x80x2 upstream region thereof (SEQ ID: 1). This 5xe2x80x2 upstream region may contain the regions regulating the LKB1 gene expression (e.g. promoters and enchancers), and a person skilled in the art could easily specify genetic regions regulating the LKB1 gene expression, using several known methods in combination. A method for specifying the region regulating gene, for example, comprises the following steps: (a) constructing a vector in which a reporter gene is joined to downstream of the 5xe2x80x2 upstream region of the LKB1 gene (DNA composed of the whole or a portion of sequence shown in SEQ ID:1); (b) introducing said vector into appropriate cells; and (c) detecting the activity of the reporter gene. Specifically, the upstream region of the LKB1 gene is cleaved into appropriate sized fragments, for example, by various restriction enzymes, and these fragments are integrated into the upstream site of a reporter gene, such as a firefly luciferase, secretory alkali phosphatase, or chloramphenicol acetyltransferase (CAT) gene to construct expression vectors (PicaGene(trademark) Vector, Wako Pure Chemicals Industries, Ltd). Subsequently, these expression vectors are introduced into appropriate host cells, such as COS, HEK293, and CHO cells, and then incubated for a certain interval. After the incubation, the intracellular and extracellular reporter gene product expression is separately measured to determine the promoter activity of the individual gene fragment integrated into the vector. Once a gene fragment showing the promoter activity is identified, such a fragment may further be cleaved into smaller fragments and subjected again to the same process as described above to define the active site to a more specified region. To finally confirm the active site, the nucleotide sequence of the region specified as the active site may be altered by, for example, site-directed mutagenesis and the activity is measured. The region regulating the LKB1 gene expression is particularly useful in the gene therapy described above, since it can direct the expression of the LKB1 gene in vivo, under natural expression control, when this region is joined to the upstream of the normal LKB1 gene described above and then administered to a patient whose LKB1 gene is mutated.
In addition, once the promoter region is defined in the upstream of the LKB1 gene, screening for compounds that can regulate the LKB1 gene expression may be easily facilitated by investigating effects on the production of the reporter gene product using reporter gene expression vectors with this site and various compounds. Such screening method comprises the steps of: (a) constructing a vector in which a reporter gene is joined to downstream of the promoter region of the LKB1 gene; (b) introducing said vector into appropriate cells; and (c) detecting the activity of the reporter gene by exposing a test compound to the said cells and/or introducing the compound into the said cells. Test compounds include, but are not limited to, proteins, peptides, synthetic compounds, natural compounds, genes, and gene products and such.
Screening for compounds that can regulate the LKB1 gene expression may be carried out by exposing a test sample to the promoter region and selecting the compound (e.g. a protein) that binds to said promoter region. For instance, transcriptional regulatory factors which control the LKB1 gene expression and bind to this promoter can be purified using affinity-purification by, creating a synthetic oligo-DNA and such containing the promoter sequence, binding this to a suitable supporting-agent, such as cellulose, and exposing it to a cell-extract and such.
Additionally, the inventors have revealed that neoplasia, such as polyps, develop as a result of a mutation in the LKB1 gene in the patients with Peutz-Jeghers syndrome. This finding lead to a notion decreasing in the amount or activity of the LKB1 protein can render normal cells temporary cell proliferation activity. Therefore, the artificial reduction in the amount or activity of the LKB1 protein, which is achieved by utilizing anti-sense DNA for the LKB1 gene or the cDNA thereof, or by utilizing a compound which inhibits the activity of the LKB1 gene, will possibly serve to treat the diseases which require fresh cell proliferation, such as wound curing and anagenesis.